Rat Cerebrocortical Slices Research Articles

13C NMR Metabolomic Evaluation of Immediate and Delayed Mild Hypothermia in Cerebrocortical Slices after Oxygen–Glucose Deprivation

Mild brain hypothermia (32°-34°C) after human neonatal asphyxia improves neurodevelopmental outcomes. Astrocytes but not neurons have pyruvate carboxylase and an acetate uptake transporter. C nuclear magnetic resonance spectroscopy of rodent brain extracts after administering [1-C]glucose and [1,2-C]acetate can distinguish metabolic differences between glia and neurons, and tricarboxylic acid cycle entry via pyruvate dehydrogenase and pyruvate carboxylase. Neonatal rat cerebrocortical slices receiving a C-acetate/glucose mixture underwent a 45-min asphyxia simulation via oxygen-glucose-deprivation followed by 6 h of recovery. Protocols in three groups of N=3 experiments were identical except for temperature management. The three temperature groups were: normothermia (37°C), hypothermia (32°C for 3.75 h beginning at oxygen--glucose deprivation start), and delayed hypothermia (32°C for 3.75 h, beginning 15 min after oxygen-glucose deprivation start). Multivariate analysis of nuclear magnetic resonance metabolite quantifications included principal component analyses and the L1-penalized regularized regression algorithm known as the least absolute shrinkage and selection operator. The most significant metabolite difference (P<0.0056) was [2-C]glutamine's higher final/control ratio for the hypothermia group (1.75±0.12) compared with ratios for the delayed (1.12±0.12) and normothermia group (0.94±0.06), implying a higher pyruvate carboxylase/pyruvate dehydrogenase ratio for glutamine formation. Least Absolute Shrinkage and Selection Operator found the most important metabolites associated with adenosine triphosphate preservation: [3,4-C]glutamate-produced via pyruvate dehydrogenase entry, [2-C]taurine-an important osmolyte and antioxidant, and phosphocreatine. Final principal component analyses scores plots suggested separate cluster formation for the hypothermia group, but with insufficient data for statistical significance. Starting mild hypothermia simultaneously with oxygen-glucose deprivation, compared with delayed starting or no hypothermia, has higher pyruvate carboxylase throughput, suggesting that better glial integrity is one important neuroprotection mechanism of earlier hypothermia.

The protective effect of propofol pretreatment on glutamate injury of neonatal rat brain slices

To study the protective effect of propofol precondition against glutamate (Glu) neurotoxicity to neonatal rat cerebrocortical slices. Brain slices of Sprague-Dawley (SD) rats were cultured in vitro and observed the morphologic changes. Brain slices were randomly divided into three groups: blank control group, Glu injury group (1 mmol/L Glu for 0.5 hour), propofol precondition group (20 mg/L propofol for 24 hours), each n=12. Changes in pathological and ultra-structure of cells were observed using microscope. Lactate dehydrogenase (LDH) leakage rate was measured. Meanwhile, the expression of glial fibrillary acidic protein (GFAP) was detected by immunohistochemical technology, then the positive cell were counted. Cultured brain slices of cell were intact and survived well. Hematoxylin-eosin (HE) staining, electron microscopy and LDH test results showed that cerebral film neuron severely damage, gliosis, edema, LDH leakage rate in Glu injury group were significantly more severe compared with blank control group [(68.5±2.0)% vs. (16.0±2.5)%, P<0.01]. Reduce the brain slice of the propofol pretreatment group of neuronal cell jury, cell shape recovery significantly reduced LDH leakage rate compared with the Glu injury group [(38.5±2.4)% vs. (68.5±2.0)%, P<0.05]. Immunohistochemical detection of GFAP expression of Glu injury group glial cell body swelling, producing increase in the number of GFAP positive reaction strong, the number of positive cells compared with blank control group was significantly increased (50±5 cells/HP vs. 10±3 cells/HP, P<0.01). The recovery of propofol pretreatment group glial cell morphology, cell processes slender GFAP positive reaction decreased the number of positive cells compared with the Glu injury group was significantly decreased (30±4 cells/HP vs. 50±5 cells/HP, P<0.05). Propofol pretreatment has protective effect against Glu injured rat cerebrocortical slices.

The Effects of Benzodiazepines on Urotensin II-Stimulated Norepinephrine Release from Rat Cerebrocortical Slices

Urotensin II (UII) and its receptor (UT) are implicated in mood disorders, such as stress and anxiety, and this may result, at least in part, from increased norepinephrine release from the cerebral cortex. Benzodiazepines have been widely used as hypnotics and anxiolytics, producing a decrease in cerebrocortical norepinephrine release. We hypothesized that there was some interaction between benzodiazepines and the UII system in the cerebral cortex. In the present study, we have examined the effects of benzodiazepines on UII-increased norepinephrine release from rat cerebrocortical slices and intracellular Ca(2+) concentrations ([Ca(2+)]i) in HEK293 cells expressing rat UT receptor (HEK293-rUT cells). Midazolam, diazepam and flunitrazepam concentration-dependently inhibited UII-evoked norepinephrine release but did not affect [Ca(2+)]i. The IC(50) of midazolam for inhibition of UII-evoked norepinephrine release (0.32 microM, P < 0.01) was significantly lower than that of diazepam (187 microM) or flunitrazepam (40 microM). The inhibitory effects of midazolam on UII-evoked norepinephrine release were significantly attenuated by flumazenil, a benzodiazepine site antagonist. The present study suggests that midazolam, at clinically relevant concentration, significantly inhibited UII-evoked norepinephrine release. This inhibitory effect may be partially mediated via central benzodiazepine receptors.

Orexin A Decreases Ketamine-Induced Anesthesia Time in the Rat: The Relevance to Brain Noradrenergic Neuronal Activity

Orexins (OXs) regulate wakefulness, and a lack of OX Type-I receptors cause narcolepsy. OX selectively increases norepinephrine (NE) release from rat cerebral cortical slices, and brain noradrenergic neurons are involved in the sleep-wakefulness cycle. Ketamine increases NE release from the rat cerebral cortex. We hypothesized that OX would affect ketamine anesthesia's interactions with brain noradrenergic neuronal activity. We used Sprague Dawley rats. We studied 1) in vivo effects of orexin A (OXA) and SB-334867-A (Orexin-1 receptor antagonist) on ketamine-induced anesthesia time, 2) in vivo effects of OXA on ketamine-induced increase in NE release from the frontal cortex assessed using microdialysis, and 3) in vitro effects of ketamine on OXA-evoked NE release from rat cerebrocortical slices. 1) Intracerebroventricular OXA 1 nmol significantly decreased ketamine anesthesia time by 20%-30% at 50, 100, and 125 mg/kg intraperitoneal (IP) ketamine. SB-334867-A fully reversed the decrease produced by OXA. 2) OXA also decreased the release of NE induced by ketamine even though OXA increased the release of NE in rat prefrontal cortex. Maximum NE release in Group OX + K (intracerebroventricular OXA 1 nmol + IP ketamine 100 mg/kg) was 271% and was significantly smaller than that in Group K (ketamine 100 mg/kg IP, 390% of baseline, P = 0.029). 3) Ketamine inhibited OX-evoked NE release with clinically relevant IC(50) values. Orexinergic neurons may be an important target for ketamine. OXA antagonized ketamine anesthesia via Orexin-1 receptor with noradrenergic neurons.

Interaction between orexinergic neurons and NMDA receptors in the control of locus coeruleus–cerebrocortical noradrenergic activity of the rat

Several studies suggest that NMDA glutamate receptors may play an important role in the activation of a number of brain regions by orexin (OX). We hypothesized that OX and NMDA receptors may interact with cerebrocortical noradrenergic neuron originating from the locus coeruleus (LC). To test this hypothesis, using rats as experimental animals, we examined (i) in vitro effects of MK801 on OXA-evoked norepinephrine release from rat cerebrocortical slices, (ii) in vivo interaction between OXA and the NMDA receptor antagonist, MK801 on norepinephrine release from the prefrontal cortex assessed using microdialysis and (iii) MK801 and OXA-modulation of the electroencephalogram (EEG). We have found that MK801 produced a concentration-dependent inhibition of OXA-evoked norepinephrine release from rat cerebrocortical slices with the IC 50 of 0.9 µM. Moreover, we have also found that icv OXA dose-dependently stimulated norepinephrine release from the rat prefrontal cortex saturating at 213% of baseline. In addition, ip MK801 0.1 mg/kg also significantly increased norepinephrine release in prefrontal cortex to 213%. However, these increases in norepinephrine release were significantly reduced by ∼ 70% by simultaneous administration of icv OXA 1 nmol and ip MK801 0.1 mg/kg. Both OXA and MK801 decreased sleep and increased wakefulness, but co-administration caused a return to base-line sleep state. These findings strongly indicate that there is a significant interaction between orexinergic neurons and NMDA receptors in the control of LC–cerebrocortical noradrenergic activity.

Fructose-1,6-bisphosphate does not preserve ATP in hypoxic–ischemic neonatal cerebrocortical slices

Fructose-1,6-bisphosphate (FBP), an endogenous intracellular metabolite in glycolysis, was found in many preclinical studies to be neuroprotective during hypoxia–ischemia (HI) when administered exogenously. We looked for HI neuroprotection from FBP in a neonatal rat brain slice model, using 14.1 T 1H/ 31P/ 13C NMR spectroscopy of perchloric acid slice extracts to ask: 1) if FBP preserves high energy phosphates during HI; and 2) if exogenous [1- 13C]FBP enters cells and is glycolytically metabolized to [3- 13C]lactate. We also asked: 3) if substantial superoxide production occurs during and after HI, thinking such might be treatable by exogenous FBP's antioxidant effects. Superfused P7 rat cerebrocortical slices (350 μm) were treated with 2 mM FBP before and during 30 min of HI, and then given 4 h of recovery with an FBP-free oxygenated superfusate. Slices were removed before HI, at the end of HI, and at 1 and 4 h after HI. FBP did not improve high energy phosphate levels or change 1H metabolite profiles. Large increases in [3- 13C]lactate were seen with 13C NMR, but the lactate fractional enrichment was always (1.1 ± 0.5)%, implying that all of lactate's 13C was natural abundance 13C, that none was from metabolism of 13C-FBP. FBP had no effect on the fluorescence of ethidium produced from superoxide oxidation of hydroethidine. Compared to control slices, ethidium fluorescence was 25% higher during HI and 50% higher at the end of recovery. Exogenous FBP did not provide protection or enter glycolysis. Its use as an antioxidant might be worth studying at higher FBP concentrations.

Urotensin II evokes neurotransmitter release from rat cerebrocortical slices

Urotensin II (UII) has been reported to modulate rapid eye movement (REM) sleep via activation of brainstem cholinergic neurons and REM sleep is regulated by locus coerleus (LC)-cerebrocortical noradrenergic neurons. We hypothesized that UII may activate LC-cerebrocortical noradrenergic neurons. To test this hypothesis, we have examined the effects of UII on norepinephrine release from rat cerebrocortical slices. In addition, the effect of the putative UT receptor antagonist [Pen(5), DTrp(7), Dab(8)]UII(4-11) (UFP-803) was assessed. We have compared this with other wakefulness-promoting neurotransmitters such as dopamine, glutamate, serotonin and histamine. We also studied the effects of UII and UFP-803 on intracellular Ca(2+) ([Ca(2+)]i) in HEK293 cells stably expressing rat UT receptor (HEK293-rUT cells). UII produced a time- (peaking at approximately 10 min following stimulation with 10nM) and concentration-dependent increase in norepinephrine release with pEC(50) and E(max) (% of basal) values of 8.78+/-0.17 (1.65 nM) and 138+/-2%, respectively. UII also evoked dopamine, serotonin and histamine release with similar pEC(50) values. UII increased glutamate release but only at high concentrations (<100 nM) and this failed to saturate. UII markedly increased [Ca(2+)](i) in HEK293-rUT cells in a concentration-dependent manner with pEC(50) of 8.26+/-0.24. The UT antagonist UFP-803 reversed both UII-increased norepinephrine release from the cerebrocortical slices (pK(B)=8.98) and [Ca(2+)](i) (pK(B)=8.87) in HEK293-rUT cells. Collectively these data suggest that UII evokes the release of norepinephrine via UT receptor activation and produces similar effects on other wakefulness-promoting neurotransmitters: these neurochemical actions of UII may be important for the control of the sleep-wake cycle.

Exogenous Ethyl Pyruvate versus Pyruvate during Metabolic Recovery after Oxidative Stress in Neonatal Rat Cerebrocortical Slices

Exogenous pyruvate and ethyl pyruvate (EP), the key ingredient in a new Ringer's solution in clinical trials, are antioxidants as well as metabolic substrates. In vivo studies show both to be protective in oxidative stress, with EP being better. The authors used an acute rat brain slice preparation to compare EP and pyruvate rescue after H(2)O(2) oxidative stress, asking whether EP was again better and whether its actions were exclusively metabolic. Oxygenated neonatal P7 cerebrocortical slices were exposed for 1 h to 2 mM H(2)O(2), and recovered for 4 h with artificial cerebrospinal fluid having 2 mM glucose and (1) 20 mM EP, (2) 20 mM pyruvate, or (3) 1 mM of the nonmetabolizable radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN). Perchloric acid extracts were studied with 31P/1H nuclear magnetic resonance at 14.1 T. Acute cell injury was assessed by counting terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL)-stained cells. At the end of recovery, preservation of adenosine triphosphate and N-acetylaspartate was better with EP than with pyruvate. Adenosine triphosphate preservation was best when PBN and EP were coadministered. 1H nuclear magnetic resonance revealed changes in lactate, alanine, gamma-aminobutyric acid, glutamate, glutamine, succinate, taurine, and myoinositol. Two-dimensional [1H-13C] heteronuclear single quantum coherence spectroscopy found that 13C-EP administration produced the same tricarboxylic acid metabolites as C-pyruvate. TUNEL-positive cell percentages with EP were less than half of those for PBN or pyruvate rescue (P < 0.05). EP enters cells, provides pyruvate as a tricarboxylic acid substrate, and is more protective. Although EP provides metabolic protection of adenosine triphosphate levels, it does not maximize antioxidant protection.

Pyruvate Improves Recovery after PARP-1-Associated Energy Failure Induced by Oxidative Stress in Neonatal Rat Cerebrocortical Slices

Previous neuron and glial cell culture studies of excessive poly (ADP-ribose) polymerase (PARP-1) activation found NAD(+) depletion, glycolytic arrest, and cell death that could be avoided by exogenous tricarboxylic acid cycle (TCA) metabolites, especially pyruvate (pyr). Pyruvate neuroprotection has been attributed to cytosolic NAD(+) replenishment, TCA metabolism, and antioxidant activity. We investigated the first two mechanisms in respiring cerebrocortical slices after a 1-h H(2)O(2) exposure to activate PARP-1. H(2)O(2) was followed by a 4-h recovery with oxy-artificial cerebrospinal fluid superfusion having either: (1) no glucose (glc) or pyruvate; (2) 10 mmol/L glc only; (3) 10 mmol/L pyruvate only; (4) both 10 mmol/L glc and 10 mmol/L pyruvate. Poly-ADP-ribosylation was quantified from Western blots and immunohistochemistry. Perchloric acid extracts were quantified with 14.1 T (31)P nuclear magnetic resonance spectroscopy. Just after H(2)O(2) exposure, ATP and NAD(+) decreased by approximately 50%, PCr decreased by 75%, and the ADP/ATP ratio approximately doubled. ATP and NAD(+) changes, but not PCr changes, were nearly eliminated if PARP inhibitors accompanied the H(2)O(2). Recovery with both pyruvate and glc was better than with glc alone, having higher ATP (0.161 versus 0.075, P<0.01) and PCr levels (0.144 versus 0.078, P<0.01), and higher viable cell counts in TUNEL and Fluoro-Jade B staining. Two-dimensional [(1)H-(13)C] HSQC spectra showed metabolism during recovery of (13)C glc or pyr. Pyruvate metabolism was primarily via pyruvate dehydrogenase, with some via pyruvate carboxylation. Pyruvate superfusion of PARP-injured brain slices helps replenish NAD(+) while providing metabolic fuel. Although this augments recovery, a strong antioxidant role for pyruvate has not been ruled out.

Lack of interaction between orexinergic and α 2-adrenergic neuronal systems in rat cerebrocortical slices

Orexinergic and norepinephrinergic α 2-adrenoceptor expressing neurons contribute to the regulation of the sleep–wakefulness cycle. In the present study, we have examined a possible interaction between orexinergic and α 2-adrenergic systems in orexin-A (100 nM)- and K + (25 mM)-evoked norepinephrine release from slices of rat cerebrocortex. In this tissue norepinephrinergic neurons are predominantly innervated via the locus coeruleus. Clonidine concentration-dependently inhibited K +-evoked norepinephrine release with pIC 50 ( I max) of 6.44 ± 0.38 (48.8 ± 6.9%). A selective orexin-1 receptor antagonist, SB-334867 was ineffective. SB-334867 concentration-dependently inhibited orexin A-evoked norepinephrine release with pIC 50 ( I max) of 6.05 ± 0.14 (86.4 ± 5.4%); clonidine (α 2-agonist) was ineffective. In contrast, yohimbine reversed the inhibitory effects of clonidine (1 μM) on K +-evoked norepinephrine release with pIC 50 ( I max) of 6.50 ± 0.34 (77.6 ± 10.9%); orexin A was ineffective. The present data suggest a lack of interaction between orexinergic and α 2-adrenergic neurons in rat cerebral cortex.

Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) is a critical regulator of energy balance at both the cellular and whole-body levels. Two upstream kinases have been reported to activate AMPK in cell-free assays, i.e., the tumor suppressor LKB1 and calmodulin-dependent protein kinase kinase. However, evidence that this is physiologically relevant currently only exists for LKB1. We now report that there is a significant basal activity and phosphorylation of AMPK in LKB1-deficient cells that can be stimulated by Ca2+ ionophores, and studies using the CaMKK inhibitor STO-609 and isoform-specific siRNAs show that CaMKKbeta is required for this effect. CaMKKbeta also activates AMPK much more rapidly than CaMKKalpha in cell-free assays. K(+)-induced depolarization in rat cerebrocortical slices, which increases intracellular Ca2+ without disturbing cellular adenine nucleotide levels, activates AMPK, and this is blocked by STO-609. Our results suggest a potential Ca(2+)-dependent neuroprotective pathway involving phosphorylation and activation of AMPK by CaMKKbeta.

P.6.004 Influence of the A1 allele of the DRD2 on alcohol craving

We have examined the effects of the recently described heptadecapeptide nocistatin on K+-evoked glutamate release from rat cerebrocortical slices in vitro. In vivo, nocistatin reverses the action of nociceptin. Nocistatin (100 nM, n=7) did not inhibit K+-evoked glutamate release alone. Nociceptin (100 nM) inhibited glutamate release by 51.7±8.3% (P<0.05, n=6) and this was fully reversed by nocistatin (100 nM). Nocistatin also appears to be an antagonist of nociceptin action in vitro.

Supraclinical concentrations of dexmedetomidine evoke norepinephrine release from rat cerebrocortical slicesPossible involvement of the orexin-1 receptor

Dexmedetomidine is a highly selective α2-agonist and reduces norepinephrine release from several neuronal tissues. However, supraclinical concentrations of dexmedetomidine have been reported to increase norepinephrine release from cardiac stores. In addition, some report using microdialysis shows that intrathecal clonidine increased norepinephrine release from the dorsal horn in mid-thoracic spinal cord but dexmedetomidine did not. Thus, in the present study we have studied effects of dexmedetomidine on norepinephrine release from rat cerebrocortical slices and compared this with clonidine. We have also used a selective α2-antagonist yohimbine and an orexin-1 receptor antagonist SB-334867 to examine whether the effects of dexmedetomidine on norepinephrine release are mediated via α2-adrenergic or orexin (OX) receptors. In addition, concentrations of orexin A in the evoked sample were also measured. Dexmedetomidine significantly increased norepinephrine release (basal = 100%) from rat cerebrocortical slices in a concentration-dependent manner with Emax 377.3 ± 8.6% and pEC50 6.12 ± 0.07, whereas clonidine significantly reduced the release with Emax 62.1 ± 6.8% and pEC50 4.55 ± 0.25. Yohimbine (10−5 M) did not affect the concentration–response curve of dexmedetomidine for norepinephrine release. However, SB-334867 concentration-dependently antagonized dexmedetomidine-evoked norepinephrine release with Imax 91.0 ± 9.4% and pIC50 5.99 ± 0.18. Orexin A concentrations did not differ between the samples. Thus, supraclinical concentrations of dexmedetomidine increase norepinephrine release from rat cerebrocortical slices, and this release may be mediated via OX1 but not α2-adrenoceptors.

Supraclinical concentrations of dexmedetomidine evoke norepinephrine release from rat cerebrocortical slices: Possible involvement of the orexin-1 receptor

Dexmedetomidine is a highly selective α 2-agonist and reduces norepinephrine release from several neuronal tissues. However, supraclinical concentrations of dexmedetomidine have been reported to increase norepinephrine release from cardiac stores. In addition, some report using microdialysis shows that intrathecal clonidine increased norepinephrine release from the dorsal horn in mid-thoracic spinal cord but dexmedetomidine did not. Thus, in the present study we have studied effects of dexmedetomidine on norepinephrine release from rat cerebrocortical slices and compared this with clonidine. We have also used a selective α 2-antagonist yohimbine and an orexin-1 receptor antagonist SB-334867 to examine whether the effects of dexmedetomidine on norepinephrine release are mediated via α 2-adrenergic or orexin (OX) receptors. In addition, concentrations of orexin A in the evoked sample were also measured. Dexmedetomidine significantly increased norepinephrine release (basal = 100%) from rat cerebrocortical slices in a concentration-dependent manner with E max 377.3 ± 8.6% and pEC 50 6.12 ± 0.07, whereas clonidine significantly reduced the release with E max 62.1 ± 6.8% and pEC 50 4.55 ± 0.25. Yohimbine (10 −5 M) did not affect the concentration–response curve of dexmedetomidine for norepinephrine release. However, SB-334867 concentration-dependently antagonized dexmedetomidine-evoked norepinephrine release with I max 91.0 ± 9.4% and pIC 50 5.99 ± 0.18. Orexin A concentrations did not differ between the samples. Thus, supraclinical concentrations of dexmedetomidine increase norepinephrine release from rat cerebrocortical slices, and this release may be mediated via OX 1 but not α 2-adrenoceptors.

Endogenous D-serine is involved in induction of neuronal death by N-methyl-D-aspartate and simulated ischemia in rat cerebrocortical slices.

Emerging evidence indicates that D-serine rather than glycine serves as an endogenous agonist at glycine site of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, in several nervous tissues, including the developing cerebellum and the retina. Here, we examined whether endogenous D-serine plays a significant role in neuronal damage resulting from excitotoxic insults in the cerebral cortex, using rat brain slices maintained in a defined salt solution. Neuronal cell death induced by application of NMDA or by oxygen-glucose deprivation (simulated ischemia) was markedly suppressed by a competitive glycine site antagonist 2,7-dichlorokynurenic acid. Addition of glycine or D-serine did not augment neuronal damage by NMDA or simulated ischemia, indicating that sufficient amount of glycine site agonist(s) is supplied endogenously within the slices. Application of D-amino acid oxidase, an enzyme that degrades D-serine, markedly inhibited neuronal damage by NMDA and simulated ischemia, which was reversed by addition of excess D-serine or glycine. Sensitivity to the glycine site antagonist of NMDA- or ischemia-induced damage was not affected by the presence of a non-NMDA receptor antagonist, suggesting that kainate receptor-stimulated D-serine release as demonstrated in primary cultured astrocytes does not contribute significantly to the extent of neuronal injury in these settings. The present results suggest that endogenous supply of D-serine as a glycine site agonist is important for neuronal injury involving NMDA receptor overactivation in the cerebral cortex.

Role of Noradrenergic and Orexinergic Neurons in General Anesthesia

Considerable evidence indicates that the GABAA receptor is a molecular target for general anesthetics in the CNS. However, most clinical anesthetists are puzzled by this (unifying) observation as the clinical features of anesthesia differ among anesthetics. Central noradrenergic neurons are known to regulate physiological functions including the sleep-wake cycle, motor activity, and autonomic nervous function. These physiological responses are depressed during general anesthesia. In addition, animal experiments have shown that general anesthetics, which activate GABAA receptors, depress brain noradrenergic neurons. Thus, we have focused on this neuronal system as a “wiring” target for general anesthetic action. However, ketamine, nitrous oxide and xenon, which act as NMDA receptor antagonists, markedly increase noradrenaline release in tissues from several brain regions. Consistent with the clinical features of anesthesia modulation of noradrenergic neurons could differ between the type of anesthetic agents. Therefore, loss of consciousness could be produced by cerebral depression but also over-excitation (e.g. convulsion). Sleep is also induced by not only hypothermia (hibernation) but also body heating that activates heat-sensitive neurons in the preoptic area. Wakefulness, that is to say, may maintain within a set range and that when this range is exceeded (above or below), unconsciousness may be induced. Recently we have accumulated several pieces of evidence supporting this hypothesis. In addition, we found that orexins, wakefulness-promoting neuropeptides, predominantly evoke noradrenaline release from rat cerebrocortical slices, and that activation of orexinergic neurons reduces the duration of anesthesia. Thus, noradrenergic and orexinergic neurons may be involved in the mechanism of general anesthesia. Keywords: noradrenergic, orexinergic neurons, anesthesia, gabaa receptor, ketamine, nmda receptor antagonists

Orexinergic neurons and barbiturate anesthesia

Orexins (OXs) regulate sleep with possible interactions with brain noradrenergic neurons. In addition, noradrenergic activity affects barbiturate anesthesia. As we have also recently reported that OXs selectively evoke norepinephrine release from rat cerebrocortical slices we hypothesized that barbiturate anesthesia may result from of an interaction with central orexinergic systems. To test this hypothesis, we performed a series of in vivo and in vitro studies in rats. In vivo, the effects of i.c.v. OX A, B and SB-334867-A (OX 1 receptor antagonist) on pentobarbital, thiopental or phenobarbital-induced anesthesia times (loss of righting reflex) was assessed. In vitro effects of barbiturates and SB-334867-A on OX-evoked norepinephrine release from cerebrocortical slice was examined. In Chinese hamster ovary cells expressing human OX 1/OX 2 receptors OX A- and B-evoked increases in intracellular Ca 2+ were measured with and without barbiturates OX A and B significantly decreased pentobarbital, thiopental and phenobarbital anesthesia times by 15–40%. SB-334867-A increased thiopental-induced anesthesia time by approximately by 40%, and reversed the decrease produced by OX A. In vitro, all anesthetic barbiturates inhibited OX-evoked norepinephrine release with clinically relevant IC 50 values. A GABA A antagonist, bicuculline, did not modify the inhibitory effects of thiopental and the GABA A agonist, muscimol, did not inhibit norepinephrine release. In addition there was no interaction of barbiturates with either OX 1 or OX 2 receptors. Collectively our data suggest that orexinergic neurons may be an important target for barbiturates, and GABA A, OX 1 and OX 2 receptors may not be involved in this interaction.

Sensitivity to selective adenosine A 1 and A 2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A 1 and A 2A receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A 1 and A 2A receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices. Slices were superfused with oxygen- and glucose-deprived medium and [ 3H] d-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca 2+ ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [ 3H] d-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor dl-TBOA prevented the ischemia-evoked efflux of [ 3H] d-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [ 3H] d-aspartate or glutamate was increased by the A 1 receptor antagonist DPCPX. The A 2A antagonist SCH 58261 decreased [ 3H] d-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC 50: 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A 1 or the A 2A receptor antagonist was found on the ischemia-evoked efflux of [ 3H] d-aspartate in Ca 2+-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A 1 and stimulatory A 2A receptors that down- or up-regulate the vesicular-like component of glutamate release.

Lack of an interaction between orexinergic and opioid/nociceptinergic systems in rat cerebrocortical slices

We have recently reported that orexins (OXs) selectively evoke norepinephrine release from rat cerebrocortical slices. In the present study, we have examined orexin–opioid interactions in OXA (100 nM) and K + (40 mM)-evoked norepinephrine release. OXA-evoked norepinephrine release was reversed ∼90% by SB-334867 (OX 1-receptor antagonist) (10 μM) but not naloxone (10 μM). [D-Pen 2,D-Pen 5]-enkephalin (DPDPE) (DOP-agonist) and nociceptin/orphanin-FQ (N/OFQ) also failed to affect OXA-evoked release. [D-Ala 2,N-Me-Phe 4,Gly 5-ol]-enkephalin (DAMGO) (MOP-agonist) and spiradoline (KOP-agonist) significantly reduced OXA-evoked release with the concentration producing 50% of the maximal inhibition (EC 50) [maximal inhibition (E max)] of 3.2 μM [41.8%] and 4.3 μM [54.9%] respectively. The effects of DAMGO and spiradoline were naloxone (10 μM)-insensitive. In contrast, naloxone significantly antagonized the inhibitory effects of DAMGO and spiradoline on K +-evoked release. We conclude that opioid receptors (DOP and KOP) are involved in K + but not OXA-evoked release. Moreover, we have failed to demonstrate an interaction between orexinergic and opioid/N/OFQ-ergic systems in this system.

Taurine counteracts cell swelling in rat cerebrocortical slices exposed to ammonia in vitro and in vivo.

Taurine is believed to fulfil a wide range of physiological functions within the mammalian body1. While it is believed that one of these may be neuroprotection, neither its relative significance in this capacity, nor the underlying mechanism, are clear. Data in favour of this concept include taurine’s ability to counteract the toxic effect of MPP+ in brain slices2 and kainite-evoked excitotoxicity in cultured neurons3 (for exhaustive review see4).